1. Field of the Invention
The present invention relates to a novel protein prepared by modifying a native antifreeze protein, and more particularly to a multimerized antifreeze protein, which exhibits excellent ice nuclei growth inhibition at low concentrations and is used as an inhibitor for ice recrystallization and as a cryopreservative, a method for producing the same, and DNA encoding a multimerized antifreeze protein.
2. Prior Art
Antifreeze peptides and antifreeze proteins (AFPs) generally have properties such as 1) thermal hysteresis, 2) inhibition of ice recrystallization, and 3) modification of ice growth. Accordingly, the use as an additive for ice creams, in which flavor and taste deteriorate due to the recrystallization caused by cold storage, or the use as a cryopreservative for cells and organs has been proposed. In a cooling system, cold storage, or the like using ice slurry, AFPs are expected as effective additives capable of dissolving blocked piping systems caused by ice recrystallization. Up to the present, native antifreeze proteins derived from plants and fishes have been mainly used in attempts to: maintain the quality of frozen foods such as ice creams; protect cells during cryopreservation; and apply for cooling systems, cold storages, or the like.
Antifreeze peptides and antifreeze proteins (AFPs), as described in more detail hereafter, generally have at least one of the following properties: 1) thermal hysteresis; 2) inhibition of ice recrystallization; or 3) modification of ice growth. Thus, the use thereof for maintaining the quality of foods or cells during cryopreservation has been proposed (Marilyn Greffith and K. Vanya Ewart, 1995. Biotechnology Advance 13: 375–402.). It has been suggested that these properties of AFP are derived from the phenomenon that binding of AFP to ice surface results in a creating convex ice surface, which energetically unfavorable for water to join the ice lattice.
AFPs have been discovered in many organisms such as fishes, insects, plants, bacteria, and fungi (John Barrett, 2001. Int J Biochem Cell Biol. 33: 105–117). One of the AFPs, which have been extensively researched, is the AFP derived from polar fishes. The fish—into five structural types; AFGP, i.e., an antifreeze glycoprotein derived AFP can be classified, which has a repeated tripeptide motif (Ala-Ala-Thr) with a disaccharide attached to threonine and is approximately 2,600 to 34,000 Da protein; Type I AFP, which is alanine-rich, α-helix protein; Type II AFP, which is cysteine-rich protein and appears to be homolog of the carbohydrate recognition domain of C-type lectins; Type III AFP, which is a small globular protein with a molecular weight of 6,500 to 7,000 Da; and Type IV AFP, which is predicted to be four-helix bundle structure. Fish-derived AFP is known to cause ice nuclei to grow into bipyramidal ice crystals (FIG. 12A), and this mechanism is construed to be as follows.
In general, when ice nuclei appear in an aqueous solution, the ice crystal first grows into a planar hexagon or square plate. The growth in a vertical direction to the plate is approximately 100 times slower than that in a planar direction. In contrast, when an antifreeze protein is present in the aqueous solution, ice crystal growth in the planar direction is inhibited, then the first-formed plate acts as a base surface upon which smaller plates are successively stacked on top of each other in the vertical direction to the base surface, and finally, the plates slowly grow into a bipyramidal ice crystal comprising two pyramidals jointed to each other.
Accordingly, when the body fluid of the fish having an antifreeze protein is cooled to subzero temperature, bipyramidal ice crystals are observed in the body fluid under a microscope. Such bipyramidal ice crystals are generated by the capacity of the antifreeze protein which specifically binds to the 12 equivalent bipyramidal planes of ice crystal. In the freezing temperature ranging at 0° C. or below, microscopically, the antifreeze proteins in the body fluid generate infinite number of bipyramidal ice crystals, which do not bind to each other. This is macroscopically observed as a non-freezing phenomenon (antifreeze activities) of specimens.
In 1995, Xin Wang et al. reported three major Type III AFPs derived from antarctic eel pout, and among them, the AFP designated as RD3 had a sequencecomprising two Type III AFPs (N-domain and C-domain) ligated to each other by a polypeptide linker constituted of nine amino acid residues (Asp-Gly-Thr-Thr-Ser-Pro-Gly-Leu-Lys) (SEQ ID NO: 19) (Xin Wang et al., 1995. Biochim. Biophys. Acta 1247: 163–172). Further, they also reported that RD3 exhibited thermal hysteresis approximately twice as much as that of other Type III AFPs on the molar concentration basis. Kazunori Miura et al. determined the RD3 structure by using NMR and reported that two predicted ice-binding planes of RD3 were located substantially on the same plane (Kazunori Miura et al., 2001, J. Biol. Chem. 276: 1304–1310). They also reported that RD3 exhibited thermal hysteresis 6 times as much as that of Type III AFP on the molar concentration basis in low molar concentrations ranging from 0.1 to 0.2 mM. This indicates that RD3 possesses thermal hysteresis activity as much as three times per molecule of Type III AFP in low concentrations.
In the past, quality maintenance of frozen foods such as ice creams and protection of cells during cryopreservation using antifreeze proteins derived from plants and fishes have been mainly attempted, although none has yet been put to practical use. Regardless of the high effectiveness being expected, the practical application thereof for cooling systems, cold storages, and the like, have not been forthcoming because of reasons such as insufficient level of activity and high protein requirements to obtain desired effects.